1. Field of the Invention
This invention relates generally to recombinant adenovirus vectors which overexpress adenovirus death proteins (ADP) and which are replication-restricted to cells expressing telomerase.
2. Description of the Related Art
Cancer is a leading cause of death in the United States and elsewhere. Depending on the type of cancer, it is typically treated with surgery, chemotherapy, and/or radiation. These treatments often fail: surgery may not remove all the cancer; some cancers are resistant to chemotherapy and radiation therapy; and chemotherapy-resistant tumors frequently develop. New therapies are necessary, to be used alone or in combination with classical techniques.
One potential therapy under active investigation is treating tumors with recombinant viral vectors expressing anti-cancer therapeutic proteins. Adenovirus-based vectors contain several characteristics that make them conceptually appealing for use in treating cancer, as well as for therapy of genetic disorders. Adenoviruses (hereinafter used interchangeably with xe2x80x9cAdsxe2x80x9d) can easily be grown in culture to high titer stocks that are stable. They have a broad host range, replicating in most human cancer cell types. Their genome can be manipulated by site-directed mutation and insertion of foreign genes expressed from foreign promoters.
The adenovirion consists of a DNA-protein core within a protein capsid (reviewed by Stewart et al., xe2x80x9cAdenovirus structure by x-ray crystallography and electron microscopy.xe2x80x9d in: The Molecular Repertoire of Adenoviruses, Doerfler, W. et al., (ed), Springer-Verlag, Heidelberg, Germany, p. 25-38). Virions bind to a specific cellular receptor, are endocytosed, and the genome is extruded from endosomes and transported to the nucleus. The genome is a linear double-stranded DNA of about 36 kbp, encoding about 36 genes (FIG. 1A). In the nucleus, the xe2x80x9cimmediate earlyxe2x80x9d E1A proteins are expressed initially, and these proteins induce expression of the xe2x80x9cdelayed earlyxe2x80x9d proteins encoded by the E1B, E2, E3, and E4 transcription units (reviewed by Shenk, T. xe2x80x9cAdenoviridae: the viruses and their replicationxe2x80x9d in: Fields Virology, Fields, B.N. et al., Lippencott-Raven, Philadelphia, p. 2111-2148). E1A proteins also induce or repress cellular genes, resulting in stimulation of the cell cycle. About 23 early proteins function to usurp the host cell and initiate viral DNA replication. Cellular protein synthesis is shut off, and the cell becomes a factory for making viral proteins. Virions assemble in the nucleus at about 1 day post infection (p.i.), and after 2-3 days the cell lyses and releases progeny virus. Cell lysis is mediated by the E3 11.6K protein, which has been renamed xe2x80x9cadenovirus death proteinxe2x80x9d (ADP) (Tollefson et al., J Virol. 70:2296-2306, 1996; Tollefson et al., Virol. 220:152-162, 1996). The term ADP as used herein in a generic sense refers collectively to ADP""s from adenoviruses such as, e.g. Ad type 1 (Ad1), Ad type 2 (Ad2), Ad type 5 (Ad5) or Ad type 6 (Ad6) all of which express homologous ADP""s with a high degree of sequence similarity.
The Ad vectors being investigated for use in anti-cancer and gene therapy are based on recombinant Ad""s that are either replication-defective or replication-competent. Typical replication-defective Ad vectors lack the E1A and E1B genes (collectively known as E1) and contain in their place an expression cassette consisting of a promoter and pre-mRNA processing signals which drive expression of a foreign gene. (See e.g. Felzmann et al., Gene Ther. 4: 1322-1329, 1997; Topf et al., Gene Ther. 5:507-513, 1998; Putzer et al., Proc. Natl. Acad. Sci. USA 94:10889-10894, 1997; Arai et al., Proc. Natl Acad. Sci. USA 94:13862-13867, 1997). These vectors are unable to replicate because they lack the E1A genes required to induce Ad gene expression and DNA replication. In addition, the E3 genes are usually deleted because they are not essential for virus replication in cultured cells.
It is recognized in the art that replication-defective Ad vectors have several characteristics that make them suboptimal for use in therapy. For example, production of replication-defective vectors requires that they be grown on a complementing cell line that provides the E1A proteins in trans. Such cell lines are fastidious, and generation of virus stocks is time-consuming and expensive. In addition, although many foreign proteins have been expressed from such vectors, the level of expression is low compared to Ad late proteins.
To address these problems, several groups have proposed using replication-competent Ad vectors for therapeutic use. Replication-competent vectors retain Ad genes essential for replication and thus, do not require complementing cell lines to replicate. Replication-competent Ad vectors lyse cells as a natural part of the life cycle of the vector. Another advantage of replication-competent Ad vectors occurs when the vector is engineered to encode and express a foreign protein. (See e.g. Lubeck et al., AIDS Res. Hum. Retroviruses 10:1443-1449, 1994). Such vectors would be expected to greatly amplify synthesis of the encoded protein in vivo as the vector replicates. For use as anti-cancer agents, replication-competent viral vectors would theoretically also be advantageous in that they should replicate and spread throughout the tumor, not just in the initially infected cells as is the case with replication-defective vectors.
Because many human tissues are permissive for Ad infection, a method should be devised to limit the replication of the virus to the target cells. To specifically target tumor cells, several research laboratories have manipulated the E1B and E1A regions of the adenovirus. For example, Onyx Pharmaceuticals recently reported on adenovirus-based anti-cancer vectors which are replication-deficient in non-neoplastic cells, but which exhibit a replication phenotype in neoplastic cells lacking functional p53 and/or retinoblastoma (pRB) tumor suppressor proteins (U.S. Pat. No. 5,677,178; Heise et al., Nature Med. 6:639-645, 1997; Bischoff et al., Science 274:373-376, 1996). This phenotype is reportedly accomplished by using recombinant adenoviruses containing a mutation in the E1B region that renders the encoded E1B-55K protein incapable of binding to p53 and/or a mutation(s) in the E1A region which make the encoded E1A protein (p289R or p243R) incapable of binding to pRB and/or p300 and/or p107. E1B-55K has at least two independent functions: it binds and inactivates the tumor suppressor protein p53, and it is required for efficient transport of Ad MRNA from the nucleus. Because these E1B and E1A viral proteins are involved in forcing cells into S-phase, which is required for replication of adenovirus DNA, and because the p53 and pRB proteins block cell cycle progression, the recombinant adenovirus vectors described by Onyx should replicate in cells defective in p53 and/or pRB, which is the case for many cancer cells, but not in cells with wild-type p53 and/or pRB. Onyx has reported that replication of an adenovirus lacking E1B-55K, named ONYX-015, was restricted to p53-minus cancer cell lines (Bischoff et al., supra), and that ONYX-015 slowed the growth or caused regression of a p53-minus human tumor growing in nude mice (Heise et al., supra). Others have challenged the Onyx report claiming that replication of ONYX-015 is independent of p53 genotype and occurs efficiently in some primary cultured human cells (Harada and Berk, J. Virol 73:5333-5344, 1999). ONYX-015 does not replicate as well as wild-type adenovirus because E1B-55K is not available to facilitate viral mRNA transport from the nucleus. Also, ONYX-015 expresses less ADP than wild-type virus.
As an extension of the ONYX-015 concept, a replication-competent adenovirus vector was designed that has the gene for E1B-55K replaced with the herpes simplex virus thymidine kinase gene (Wilder et al., Gene Therapy 6:57-62, 1999). The group that constructed this vector reported that the combination of the vector plus gancyclovir showed a therapeutic effect on a human colon cancer in a nude mouse model (Wilder et al., Cancer Res. 59:410-413, 1999). However, this vector lacks the gene for ADP, and accordingly, the vector will lyse cells and spread from cell-to-cell less efficiently than an equivalent vector that expresses ADP.
To target tumor cells, other research groups have taken advantage of the differential expression of telomerase in dividing cells. Telomerase is a ribonucleoprotein enzyme which is responsible for the maintenance of telomeres. Telomeres are long tandem repetitions of a simple sequence, for example TTAGGG, at both ends of the chromosomes, the very ends of which, because of the nature of DNA replication do not get duplicated (review Blackburn, E. H. Nature 350:569-573, 1991). As a result, telomeres shorten by each round of cell division (Harley, C B. et al. Nature 345:458-460, 1990), in the long run causing chromosomal instability and cellular senescence (Greider, C. W. Cell 67:645-647, 1991). To counteract this effect, embryonic cells, germ cells, stem cells, and hematopoietic cells (Ulaner, G/A. et al. Cancer Res. 58:4168-4172, 1998; Broccoli, D. et al. Proc. Natl. Acad. Sci. USA 92, 9082-9086, 1995; Kalquist, K. A. et al. Nature Genetics 19:182-186, 1998) produce the telomerase enzyme, which maintains the original length of the telomeres. Some epithelial basal cells in the skin and intestine also express low levels of telomerase (Yasumoto, S. et al. Oncogene 13:433-439,1996; Hxc3xa4rle-Bachor, C. and Boukamp, P. Proc. Natl. Acad. Sci. USA 93:6476-6481, 1996). Cancerous cells, on the other hand, are continuously dividing, without going into senescence. After an initial period of shortening in these dividing cells, the lengths of the telomeres stabilize. Further research proved that this stabilization was due to the reactivation of telomerase (Blasco, M. A. et al. Nature Genetics 12:200-204, 1996). A systematic search showed that late stage tumors exhibit high levels of telomerase activity (Kim, N. W. et al. Science 266:2011-2015, 1994; Shay, J. W. and Bachetti, S. Eur. J. Cancer, 33:787-791, 1997; Yan, P. et al. Cancer Res. 59:3166-3170, 1999).
The telomerase holoenzyme consists of two subunits: an RNA molecule and a protein (Morin, G. B. Cell 59:521-529, 1989). The RNA serves as the template for the telomere sequences (Feng, J. et al. Science 269:1236-1241, 1995), while the protein (human Telomerase Reverse Transcriptase or xe2x80x9chTERTxe2x80x9d) harbors reverse transcriptase activity (Harrington, L. et al. Science 275:973-977, 1997). The expression of hTERT is tightly regulated (Meyerson, M. et al. Cell 90:785-789, 1997). The regulatory role of hTERT in telomerase activity is further evidenced by the fact that introducing hTERT into telomerase-negative cells results in telomerase activation (Bodnar, A. G. et al. Science 279:349-352, 1998).
The regulation occurs mainly at the transcriptional level, though other mechanisms-alternative splicing (Ulaner, G. A. et al. Int. J Cancer 85:330-335, 2000)xe2x80x94have been implicated, too. The promoter of hTERT was cloned and sequenced by several groups (Cong, Y-S. et al Hum. Mol. Genet. 8:137-142, 1999, Horikawa, I. et al. Cancer Res. 59:826-830, 1999; Takakura, M. et al Cancer Res. 59:551-559, 1999; Wick, M. et al. Gene 232:97-106, 1999). It was shown that the isolated hTERT promoter was unable to direct the transcription of a reporter gene in cells with no telomerase activity, but it worked effectively in established telomerase positive cell lines (Horikawa et al.). The hTERT promoter binds various transcription factors (review Poole, J. C. et al. Gene 269:1-12, 2001). Of these, the cMyc/Max/Mad1 factors seem to be the most important for regulation. The promoter contains two binding sites (E-boxes) that can bind either the cMyc/Max or the Mad 1/Max heterodimer. While the former one activates, the latter represses hTERT transcription. The distribution of cMyc and Mad1 in adult organs/tissues coincides with the activity xe2x80x94or lack of activityxe2x80x94 of the hTERT promoter in those tissues (Gxc3xcnes, C. et al. Cancer Res. 60:2116-2121, 2000). In certain organs (endometrium, ovary) estrogen (Misiti, S. et al. Molec. Cell. Biol. 20:3764-3771, 2000), in others (kidney, spleen) the Wilms"" Tumor 1 tumor suppressor (WT1) (Oh, S. et al. J. Biol. Chem. 174:37473-37478, 1999), might have a regulatory role.
By using the hTERT promoter, any protein can be expressed selectively in telomerase positive, that isxe2x80x94at least in adult human-neoplastic cells. Researchers have expressed pro-apoptotic proteins (Koga, S. et al. Hum. Gene. Ther. 11: 1397-1406, 2000), bacterial toxins (Abduol-Ghani, R. et al. Molecular Therapy 2:539-544, 2000), and prodrug converting enzymes (Majumdar, A. S. et al. Gene Therapy 8:568-578, 2001) using various hTERT promoter containing vectors. Though limited to telomerase positive cells, these expression vectors do not effectively deliver the anti-cancer agent to neighboring tumor cells. Instead, to be effective, the vector would need to be introduced into each tumor cell.
Thus, there is a continuing need for an efficient and effective anti-cancer adenovirus vector that could specifically target neoplastic cells, while replicating poorly or not at all in normal tissue, and efficiently spreading to neighboring neoplastic cells, thereby maximizing the cancer-killing ability of the adenovirus vector.
Briefly, therefore, the present invention is directed to novel adenovirus vectors which overexpress an adenovirus death protein (xe2x80x9cADPxe2x80x9d) and which are replication-restricted to cells expressing telomerase. Overexpression of ADP by a recombinant adenovirus allows the construction of a replication-competent adenovirus that kills cells expressing telomerase and spreads from cell-to-cell at a rate similar to or faster than that exhibited by adenoviruses expressing wild-type levels of ADP, even when the recombinant adenovirus contains a mutation that would otherwise reduce its replication rate in non-neoplastic cells. The work herein demonstrates that substitution of the human telomerase reverse transcriptase promoter (xe2x80x9chTERTxe2x80x9d) for the adenovirus E4 promoter allows restriction of replication of the adenovirus to cells expressing telomerase without the need for complementation to achieve replication competence in these cells.
In one embodiment of the invention, the recombinant adenovirus vector comprises an ADP gene, a hTERT promoter, and at least one mutation in the E3 region. In a preferred embodiment, the hTERT-ADP-expressing vector comprises a recombinant adenovirus vector lacking expression of at least one E3 protein selected from the group consisting of: gp19K; RIDxcex1 (also known as 10.4K); RIDxcex2 (also known as 14.5K) and 14.7K. Because wild-type E3 proteins inhibit immune-mediated inflammation and/or apoptosis of Ad-infected cells, it is believed that a recombinant adenovirus lacking one or more of these E3 proteins will stimulate infiltration of inflammatory and immune cells into a tumor treated with the adenovirus and that this host immune response will aid in destruction of the tumor as well as tumors that have metastasized. A mutation in the E3 region would impair its wild-type function, making the viral-infected host susceptible to attack by the host""s immune system. Such a vector is identified as GZ3-TERT and its sequence is represented in SEQ ID NO. 1.
In still another embodiment of the invention, the recombinant adenovirus vector comprises an ADP gene, a hTERT promoter, and at least one inactivating mutation in the E1A region, resulting in a loss of transformation of resting cells by E1A without inhibiting its function in the targeted cells. The mutation in the E1A region disrupts E1A protein binding to p300 and/or members of the Rb family. Without this binding, E1A cannot provoke G0 exit and subsequent entry into S phase, an essential progression for synthesis of molecules necessary for adenoviral replication. Thus, a resting cell (i.e. one in G0 state) cannot support replication of the recombinant adenovirus vector of the present invention. Such a vector is identified as KD3-TERT and its sequence is represented in SEQ ID NO. 2.
Another embodiment of the invention involves a pharmaceutical composition comprising the recombinant adenovirus vector of the present invention in association with a pharmaceutically acceptable carrier.
Yet another embodiment of the invention is directed at an in vitro method and kit for promoting death of cells expressing telomerase comprising contacting said cell with an effective amount of KD3-TERT, GZ3-TERT, or both.
Still another embodiment of the invention is directed at a method for promoting death of neoplastic cells in a patient comprising administering an effective amount of the adenovirus vector(s) of the present invention to said patient.
Among the several advantages found to be achieved by the present invention, therefore, may be noted the provision of replication-competent adenovirus vectors, which rapidly kill cancer cells and spread from cell-to-cell in a tumor; the provision of such vectors whose replication can be restricted to cells expressing telomerase; the provision of pharmaceutical compositions for anti-cancer therapy which cause little to no side effects in normal tissues; and the provision for methods and kits for promoting death in cells expressing telomerase, such as neoplastic cells.